Clin Exp Otorhinolaryngol.  2022 May;15(2):153-159. 10.21053/ceo.2021.00857.

Inflammatory Monocytes Infiltrate the Spiral Ligament and Migrate to the Basilar Membrane After Noise Exposure

• Shin SH
• Yoo JE
• Jung J
• Choi JY
• Bae SH

• KMID: 2530161
• DOI: http://doi.org/10.21053/ceo.2021.00857

Abstract

Objectives
. Noise exposure leads to an increase in the macrophage population. This increment is thought to be caused by the transformation of infiltrated monocytes into macrophages rather than by proliferation of the cochlear resident macrophages. However, studies on infiltrated monocytes in the cochlea are scarce. Thus, we aimed to investigate the infiltration of monocytes and their transformation into macrophages after noise exposure.
Methods
. In wild-type and CX3CR1^+/GFP C57/B6 mice, inflammatory monocytes were identified by immunofluorescence of mouse cochlear cells. The findings were confirmed and quantitated by flow cytometry.
Results
. One day after noise exposure, monocytes were identified in the spiral ligament. Flow cytometric analysis confirmed that the monocyte population peaked on post-noise exposure day 1 and decreased thereafter. On day 3 after noise exposure, amoeboid-type macrophages increased in the crista basilaris, and on day 5, they spread to the basilar membrane.
Conclusion
. Infiltrated monocytes were successfully observed 1 day after noise exposure, preceding the increase in the macrophage population. This finding supports the proposal that infiltrated monocytes transform into macrophages.

Keyword

Cochlea; Macrophages; Monocytes; Noise

Figure

• 

  Fig. 1. Permanent threshold shift. Mice were exposed to 120-dB white noise for 1 hour. The findings 2 weeks after noise exposure are shown. (A) Auditory brainstem response threshold shift. (B) The basal turn of the unexposed cochlea shows preserved inner and outer hair cells. (C) The noise-exposed cochlea shows degenerated outer hair cells and intact inner hair cells. The loss of nuclei (DAPI) and cell bodies (Myo7A) indicates degenerated outer hair cells. Only a cuticular plate is observed. Green, phalloidin; Blue, DAPI; Red, Myo7A. Scale bar=20 μm.

• 

  Fig. 2. Expression of monocyte markers in the basal turn of the cochlea. The lateral cochlear wall shows (A) CX3CR1+ resident macrophages before noise exposure and (B) a significant increase in Ly6Cpositive monocytes 1 day after noise exposure. (C) Three days after noise exposure, the cochlea displayed a decrease in Ly6C-positive monocytes and an increase in CX3CR1+ macrophages. The cochlea (D) 5, and (E) 7 days after noise exposure. The dim white color indicates non-specific staining of Ly6C to fibrocytes, and the weak white color with a hollow center indicates blood vessel endothelium, which is also known to express Ly6C. In all of the images, the top is the lateral wall and the bottom is the basilar membrane. Dotted line indicates Ly6C-stained endothelial cells of the blood vessel that are confirmed by exploration of z-stack images. Green, CX3CR1; White, Ly6C. Scale bar=100 μm.

• 

  Fig. 3. Flow cytometric evaluation of the infiltrated monocytes after noise exposure. (A) Ly6C (white) and DAPI (blue) staining of the infiltrated monocytes in the spiral ligament after noise exposure indicates that Ly6C staining is not an artifact or background noise. Green, CX3CR1; White, Ly6C; and Blue, DAPI. Scale bar=20 μm. (B) The percentage of monocytes (CD11b+ , Ly6G– , Ly6Chigh) from the parent population (CD11b+ , Ly6G– ) (**P<0.01 by one-way analysis of variance with the Tukey post-hoc test). (C) The absolute cell count for CD11b+ /Ly6G– /Ly6C– cells and monocytes (CD11b+ , Ly6G– , Ly6Chigh). (D) Gating for CD11b (myelocyte lineage) and Ly6G (neutrophils) to visualize monocyte infiltration. Ly6G+ cells were excluded because they were neutrophils. N1d, 1 day after noise exposure; N3d, 3 days after noise exposure; N5d, 5 days after noise exposure; WT, wild type; SSC, side scatter; FSC, forward scatter; SSC-A, side scatter area; SSC-W, side scatter width.

• 

  Fig. 4. Migration of macrophages after acoustic injury. Whole mounted images of the basilar membrane using a confocal microscope at each time point are presented. Several CX3CR1+ resident macrophages are seen beneath the basilar membrane in (A) unexposed cochlea and (B) 1 day after noise exposure. (C) Three days postnoise exposure, an increase in CX3CR1+ cells with amoeboid morphology was seen in the junction of the basilar membrane and lateral wall (crista basilaris). Macrophages spread to the basilar membrane (D) 5, and (E) 7 days after noise exposure. All images show the lateral wall at the top and the osseous spiral lamina at the bottom. Arrowheads indicate representative amoeboid macrophages. Dotted lines indicate the borders of the basilar membrane. Green, CX3CR1; Red, F4/80; Scale bar=100 μm.

• 

  Fig. 5. Quantification of basilar membrane macrophages from four samples. Amoeboid macrophages were defined as dense, round cells without dendrites. Mature macrophages were defined as long, irregular shapes with dendrites. Cells in the basal turn (up to 60% of the distance from the apex) were counted. Two-way analysis of variance with the post-hoc Dunnett comparison was used to evaluate cell count differences from control cochlea. CTR, untreated cochlea; N1d, 1 day after noise exposure; N3d, 3 days after noise exposure; N5d, 5 days after noise exposure; N7d, 7 days after noise exposure. **P<0.01, ***P<0.001.

Reference

1. Wang Y, Hirose K, Liberman MC. Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J Assoc Res Otolaryngol. 2002; Sep. 3(3):248–68.

2. Hirose K, Discolo CM, Keasler JR, Ransohoff R. Mononuclear phagocytes migrate into the murine cochlea after acoustic trauma. J Comp Neurol. 2005; Aug. 489(2):180–94.

3. Lang H, Ebihara Y, Schmiedt RA, Minamiguchi H, Zhou D, Smythe N, et al. Contribution of bone marrow hematopoietic stem cells to adult mouse inner ear: mesenchymal cells and fibrocytes. J Comp Neurol. 2006; May. 496(2):187–201.

4. Yang W, Vethanayagam RR, Dong Y, Cai Q, Hu BH. Activation of the antigen presentation function of mononuclear phagocyte populations associated with the basilar membrane of the cochlea after acoustic overstimulation. Neuroscience. 2015; Sep. 303:1–15.

5. He W, Yu J, Sun Y, Kong W. Macrophages in noise-exposed cochlea: changes, regulation and the potential role. Aging Dis. 2020; Feb. 11(1):191–9.

6. Okano T, Nakagawa T, Kita T, Kada S, Yoshimoto M, Nakahata T, et al. Bone marrow-derived cells expressing Iba1 are constitutively present as resident tissue macrophages in the mouse cochlea. J Neurosci Res. 2008; Jun. 86(8):1758–67.

7. Wood MB, Zuo J. The contribution of immune infiltrates to ototoxicity and cochlear hair cell loss. Front Cell Neurosci. 2017; Apr. 11:106.

8. Hu BH, Zhang C, Frye MD. Immune cells and non-immune cells with immune function in mammalian cochleae. Hear Res. 2018; May. 362:14–24.

9. Auffray C, Sieweke MH, Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Annu Rev Immunol. 2009; 27:669–92.

10. Gallagher KA, Joshi A, Carson WF, Schaller M, Allen R, Mukerjee S, et al. Epigenetic changes in bone marrow progenitor cells influence the inflammatory phenotype and alter wound healing in type 2 diabetes. Diabetes. 2015; Apr. 64(4):1420–30.

11. Willenborg S, Lucas T, van Loo G, Knipper JA, Krieg T, Haase I, et al. CCR2 recruits an inflammatory macrophage subpopulation critical for angiogenesis in tissue repair. Blood. 2012; Jul. 120(3):613–25.

12. Wynn TA, Vannella KM. Macrophages in tissue repair, regeneration, and fibrosis. Immunity. 2016; Mar. 44(3):450–62.

13. Kaur T, Zamani D, Tong L, Rubel EW, Ohlemiller KK, Hirose K, et al. Fractalkine signaling regulates macrophage recruitment into the cochlea and promotes the survival of spiral ganglion neurons after selective hair cell lesion. J Neurosci. 2015; Nov. 35(45):15050–61.

14. Tan WJ, Thorne PR, Vlajkovic SM. Characterisation of cochlear inflammation in mice following acute and chronic noise exposure. Histochem Cell Biol. 2016; Aug. 146(2):219–30.

15. Bae SH, Kwak SH, Yoo JE, Kim KM, Hyun YM, Choi JY, et al. Threedimensional distribution of cochlear macrophages in the lateral wall of cleared cochlea. Clin Exp Otorhinolaryngol. 2021; May. 14(2):179–84.

16. Italiani P, Boraschi D. From monocytes to M1/M2 macrophages: phenotypical vs. functional differentiation. Front Immunol. 2014; Oct. 5:514.

17. Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T, Figueiredo JL, et al. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 2007; Nov. 204(12):3037–47.

18. Dunay IR, Damatta RA, Fux B, Presti R, Greco S, Colonna M, et al. Gr1(+) inflammatory monocytes are required for mucosal resistance to the pathogen Toxoplasma gondii. Immunity. 2008; Aug. 29(2):306–17.

19. Kimball AS, Joshi A, Carson WF 4th, Boniakowski AE, Schaller M, Allen R, et al. The histone methyltransferase MLL1 directs macrophage-mediated inflammation in wound healing and is altered in a murine model of obesity and type 2 diabetes. Diabetes. 2017; Sep. 66(9):2459–71.

20. Nahrendorf M, Pittet MJ, Swirski FK. Monocytes: protagonists of infarct inflammation and repair after myocardial infarction. Circulation. 2010; Jun. 121(22):2437–45.

21. Rose S, Misharin A, Perlman H. A novel Ly6C/Ly6G-based strategy to analyze the mouse splenic myeloid compartment. Cytometry A. 2012; Apr. 81(4):343–50.

22. Kimball A, Schaller M, Joshi A, Davis FM, denDekker A, Boniakowski A, et al. Ly6CHi blood monocyte/macrophage drive chronic inflammation and impair wound healing in diabetes mellitus. Arterioscler Thromb Vasc Biol. 2018; May. 38(5):1102–14.

23. Ghasemlou N, Chiu IM, Julien JP, Woolf CJ. CD11b+Ly6G- myeloid cells mediate mechanical inflammatory pain hypersensitivity. Proc Natl Acad Sci U S A. 2015; Dec. 112(49):E6808–17.

24. Loken MR, Brosnan JM, Bach BA, Ault KA. Establishing optimal lymphocyte gates for immunophenotyping by flow cytometry. Cytometry. 1990; 11(4):453–9.

25. Liyanage SE, Gardner PJ, Ribeiro J, Cristante E, Sampson RD, Luhmann UF, et al. Flow cytometric analysis of inflammatory and resident myeloid populations in mouse ocular inflammatory models. Exp Eye Res. 2016; Oct. 151:160–70.

26. Sato E, Shick HE, Ransohoff RM, Hirose K. Repopulation of cochlear macrophages in murine hematopoietic progenitor cell chimeras: the role of CX3CR1. J Comp Neurol. 2008; Feb. 506(6):930–42.

27. Shi X. Resident macrophages in the cochlear blood-labyrinth barrier and their renewal via migration of bone-marrow-derived cells. Cell Tissue Res. 2010; Oct. 342(1):21–30.

28. Rai V, Wood MB, Feng H, Schabla NM, Tu S, Zuo J. The immune response after noise damage in the cochlea is characterized by a heterogeneous mix of adaptive and innate immune cells. Sci Rep. 2020; Sep. 10(1):15167.